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Oxalate decarboxylase from Collybia velutips: molecular cloning and its over expression to confer resistance to fungal infection in transgenic tobacco and tomato
(The American Society for Biochemistry and Molecular Biology, Inc., 2000) Kesarwani, Meenu; Azam, Mohammad; Natarajan, K; Mehta, Anuradha; Datta, Asis
Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mecha- nism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5؅-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (Mehta, A., and Datta, A. (1991) J. Biol. Chem. 266, 23548 –23553). By screening a genomic library from Collybia velutipes with this cDNA as a probe, a genomic clone has been isolated. Sequence analyses and comparison of the genomic sequence with the cDNA sequence revealed that the cDNA is interrupted with 17 small introns. The cDNA has been successfully expressed in cytosol and vacuole of transgenic tobacco and tomato plants. The transgenic plants show normal phenotype, and the transferred trait is stably inherited to the next generation. The recombinant enzyme is partially glycosylated and shows oxalate decarboxylase activity in vitro as well as in vivo. Transgenic tobacco and tomato plants ex- pressing oxalate decarboxylase show remarkable resistance to phytopathogenic fungus Sclerotinia sclerotio- rum that utilizes oxalic acid during infestation. The result presented in the paper represents a novel approach to develop transgenic plants resistant to fungal infection.
Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus
(Proceedings of the National Academy of Sciences of the United States of America, 2000) Chakraborty, Subhra; Chakraborty, Niranjan
Improvement of nutritive value of crop plants, in particular the amino acid composition, has been a major long-term goal of plant breeding programs. Toward this end, we reported earlier the cloning of the seed albumin gene AmA1 from Amaranthus hypo- chondriacus. The AmA1 protein is nonallergenic in nature and is rich in all essential amino acids, and the composition corresponds well with the World Health Organization standards for optimal human nutrition. In an attempt to improve the nutritional value of potato, the AmA1 coding sequence was successfully introduced and expressed in tuber-specific and constitutive manner. There was a striking increase in the growth and production of tubers in transgenic populations and also of the total protein content with an increase in most essential amino acids. The expressed protein was localized in the cytoplasm as well as in the vacuole of transgenic tubers. Thus we have been able to use a seed albumin gene with a well-balanced amino acid composition as a donor protein to develop a transgenic crop plant. The results document, in addition to successful nutritional improvement of potato tubers, the feasibility of genetically modifying other crop plants with novel seed protein composition.
Attenuation of virulence and changes in morphology in Candida albicans by disruption of the N-acetylglucosamine catabolic pathway
(American Society for Microbiology, 2001) Singh, Praveen; Ghosh, Sharmistha; Datta, Asis
A Candida albicans mutant with mutations in the N-acetylglucosamine (GlcNAc) catabolic pathway gene cluster, including the GlcNAc-6-phosphate deacetylase (DAC1), glucosamine-6-phosphate deaminase (NAG1), and GlcNAc kinase (HXK1) genes, was not able to grow on amino sugars, exhibited highly attenuated virulence in a murine systemic candidiasis model, and was less adherent to human buccal epithelial cells in vitro. No germ tubes were formed by the mutant after induction with GlcNAc, but the mutant exhibited hyperfilamen- tation under stress-induced filamentation conditions. In addition, the GlcNAc catabolic pathway played a vital role in determining the colony phenotype. Our results imply that this pathway is very important because of its diverse links with pathways involved in virulence and morphogenesis of the organism.
A secretion signal is present in the Collybia velutipes oxalate decatboxylase gene
(Elsevier B.V., 2001) Azam, Mohammad; Kesarwani, Meenu; Natarajan, Krishnamurthy; Datta, Asis
The oxalate decarboxylase (OXDC) gene from Collybia velutipes is overexpressed as an active form in Schizosaccharomyces pombe. The recombinant enzyme shows similar pH optima and stability, while substrate kinetic analysis shows a ninefold decrease in K m value with respect to native OXDC. Most of the expressed protein was present in periplasm and remained firmly bound to cell-wall materials. However, 20% of enzyme expressed was secreted out into the medium suggesting the presence of a secretion signal (C. velutipes) in the oxalate decarboxylase gene. This secretion signal is associated with the N-terminal of OXDC as is evident by secretion of nonsecretory genes AmA1 and Beta-galactosidase. An expression vector using this signal is constructed for expression and secretion of heterologous proteins in S. pombe.
Role of LLD, a new locus for leaflet/pinna morphogenesis in Pisum sativum
(Indian Academy of Sciences, 2001) Prajapati, Seema; Kumar, Sushil
Properties of a mutant at the LLD (LEAF-LET DEVELOPMENT) locus in pea Pisum sativum L. are reported in this paper. Plants homozygous for the Mendelian recessive mutation lld bear leaves in which a few to many leaflets are incompletely developed. Opposite pinnae of rachis nodes often formed fused incompletely developed leaflets. The lld mutation was observed to abort pinna development at almost all morphogenetic stages. The lld mutation demonstrated high penetrance and low expressivity. The phenotypes of lld plants in tl, tac, tl tac, tl af and tl af tac backgrounds suggested that LLD function is involved in the separation of lateral adjacent blastozones differentiated on primary, secondary and tertiary rachides and lamina development in leaflets. The aborted development of tendrils and leaflets in lld mutants was related to deficiency in vascular tissue growth. The morphological and anatomical features of the leaflets formed on a tl lld double mutant permitted a model of basipetal leaflet development. The key steps of leaflet morphogenesis include origin of the lamina by splitting of a radially symmetrical growing pinna having abaxial outer surface, opposite to the vascular cylinder, through an invaginational groove, differentiation of adaxial surface along the outer boundary of split tissue in the groove and expansion of the lamina ridges so formed into lamina spans.
Involvement of plasmid in degradation of pentachlorophenol by Pseudomonoas sp. from a chemostat
(Elsevier B.V., 2001) Thakur, Indu Shekhar; Verma, Praveen K.; Upadhaya, Kailash C.
Pseudomonas sp. strain IST103 obtained from a stable bacterial consortium was capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in a chemostat. The degradation of PCP by bacterial strain proceeded through an oxidative route as indicated by accumulation of tetrachlorop-hydroquinone and chlorohydroquinone determined by high performance liquid chromatography (HPLC), and chloride molecules released in culture medium. Two different molecular size plasmids, of approximately 80 and 4 kilobase, were found to be responsible for carrying genes for degradation of PCP. This was evidenced by mutants produced by curing of plasmid by treatment of ethidium bromide. The derivatives were not able to utilize PCP, however, transformation of low molecular size plasmid of Pseudomonas sp. strain 103 into E. coli JM109 utilized PCP, indicated a possible involvement of plasmid in degradation of pentachlorophenol
The floral androcarpel organ (ACO) mutation permits high alkaloid yields in opium poppy Papaver somniferum
(Indian Academy of Sciences, 2001) Prajapati, Seema; Bajpai, Suphla; Gupta, MM; Kumar, Sushil
Isolation of a recessive aco (androcarpel organ) mutation in opium poppy Papaver somniferum, is described. The aco (aco aco) mutant plants form androcarpels in place of inner whorl stamens in their flowers. The abnormal flowers are self-fertile, as stamens in the outer whorl are normal. In aco mutant the calyx, corolla and syncarpous gynoecium are formed like in the wild type ACO plants. The androcarpel walls of aco plants synthesize and accumulate alkaloids like the main carpel walls. The aco mutation provides a means for improving carpel wall husk mass and thereby the yield of alkaloids
Level of endogenous free amino acids during various stages of culture of Vigna mungo (L.) Hepper - somatic embryogenesis, organogenesis and plant regeneration
(Indian Academy of Sciences, 2002) Sen, Jayanti; Kalia, Sanjeev; Guha-Mukherjee, Sipra
A comparative study on endogenous free amino acid levels was conducted in embryogenic and regenerating tissues vis-à-vis regenerated shoots of Vigna mungo PS1. A number of important amino acids were found to increase during initiation of embryogenesis and regeneration. To study the effects of exogenous application of amino acids in this system, media were supplemented with three amino acids, tryptophan (Trp), serine (Ser) and proline (Pro). We found that in the presence of proline, a stress associated amino acid, there was an enhancement in shoot-bud regeneration as well as in the production of somatic embryos. We also analysed the endogenous levels of amino acids in these tissues. This communication thus deals with the induction of globular embryo by auxin and its promotion by proline in V. mungo.
Inter-relationships between morphine and codeine in the Indian genetic resources of opium poppy Papaver Somniferum
(Taylor & Francis Group, 2002) Bajpai, Suphla; Gupta, Ajay Prakash; Gupta, MM; Kumar, Sushil
A set of 208 Indian and two Thai germplasm accessions of opium poppy Papaver somniferum were assessed for variation in 17 morphological characters, seed yield and content and yield of morphine from capsules and peduncles. The germplasm was found to be highly variable for all the characters evaluated. In the harvested peduncles and capsules, 13% was peduncle straw, 61% seeds and the rest capsule husk. The peduncle and capsule straw yields ranged between 0.6–2.2 and 1.4–5.3 g plant-1, respectively. Morphine content in the peduncle varied between about 0.001–0.24% and that in the capsule from 0.02 to 1.05%. On average basis morphine content in the capsule husk was more than 9-fold higher than the peduncle straw. The plant morphine yields from peduncles and capsules ranged between 1.2 and 28.6 mg plant-1. Four accessions yielded more than 20 mg of morphine plant-1. Among these, in one of the accessions about 13% of the morphine was contributed by the peduncle. The plants of high morphine yielding accessions were generally small in height, and bore white flowers and large sized ungrooved capsules with a small number of seeds, on a large peduncle.
Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat
(Elsevier B.V., 2002) Thakur, Indu Shekhar; Verma, Praveen K.; Upadhayaya, Kailash
Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatogra- phy of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS–polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1(alpha). The recombinant clones having higher potentiality to de- grade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.
Plant pre-mRNA splicing in fission yeast, Schizosaccharomyces pombe
(Academic Press, 2002) Sarmah, Bhaskarjyoti; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis
Pre-mRNA splicing or the removal of introns from precursor messenger RNAs depends on the accurate recognition of intron sequences by the splicing machinery. We have analyzed various aspects of intron sequence and structure in relation to splice site selection and splicing efficiency of a plant gene AmA1 in Schizosaccharomyces pombe. Earlier, we reported the cloning of AmA1, a seed albumin gene from Amaranthus hypochondriacus [A. Raina, A. Datta, Proc. Natl. Acad. Sci. USA 89 (1992) 11774]. In the absence of an in vitro splicing system for plants, the expression of AmA1 genomic clone in S. pombe has been used to analyze splicing of intron constructs. We aim to focus on S. pombe as a possible alternative and examined its effectiveness as a host for plant gene splicing. The results show here that pre-mRNA transcripts of AmA1 gene underwent splicing in S. pombe. Ó 2002 Elsevier Science (USA). All rights reserved.
Alkaloid profiles of the Indian land races of the opium poppy Papaver somniferum L
(Kluwer Academic Publishers, 2002) Prajapati, Seema; Bajpai, Suphla; Singh, Digvijay; Luthra, Rajesh; Gupta, Madan M.; Kumar, Sushil
The capsule husks of a set of 115 Indian land races of opium poppy Papaver somniferum L. obtained over two cropping seasons were screened for the percent contents of papaverine, reticuline, narcotine, thebaine, codeinone, codeine, morphine and oripavine. Among the accessions screened 36, 7 and 1 did not accumulate papaverine, papaverine and narcotine and codeine and morphine, respectively. The alkaloid profiles and correlations between alkaloids in all the four classes of accessions showed that in the Indian genetic resources of P. somniferum (a) morphine is synthesized from codeine rather than oripavine, (b) net alkaloid contents are low under narcotine deficiency, and (c) accumulation of morphine and codeine was limited upstream of codeinone and morphinone. The accessions identified to be harboring genetic blocks in the phenenthrine and benzylisoquinoline biosynthetic pathways will be useful for understanding the genetic control of secondary metabolism in opium poppy.
Premature termination of RNA polymerase II mediated transcription of a seed protein gene in Schizosaccharomyces pombe
(Oxford University Press, 2002) Chakraborty, Subhra; Sarmah, Bhaskarjyoti; Chakraborty, Niranjan; Datta, Asis
The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription. We show that an intronic sequence derived from the plant seed protein gene (AmA1 ) specifically acts as a transcriptional terminator in the fission yeast, Schizosaccharomyces pombe. The 3′-end points of mRNA encoded by the AmA1 gene were mapped at different positions in S.pombe and in native cells of Amaranthus hypochondriacus. Deletion analyses of the AmA1 intronic sequence revealed that multiple elements essential for proper transcriptional termination in S.pombe include two site-determining elements (SDEs) and three downstream sequence elements. RT–PCR analyses detected transcripts up to the second SDE. This is the first report showing that the highly conserved mammalian poly(A) signal, AAUAAA, is also functional in S.pombe. The poly(A) site was determined as Y(A) both in native and heterologous systems but at different positions. Deletion of these cis-elements abolished 3′-end processing in S.pombe and a single point mutation in this motif reduced the activity by 70% while enhancing activity at downstream SDE. These results indicate that the bipartite sequence elements as signals for 3′-end processing in fission yeast act in tandem with other cis-acting elements. A comparison of these elements in the AmA1 intron that function as a transcriptional terminator in fission yeast with that of its native genes showed that both require an AT-rich distal and proximal upstream element. However, these sequences are not identical. Transcription run-on analysis indicates that elongating RNA polymerase II molecules accumulate over these pause signals, maximal at 611–949 nt. Furthermore, we demonstrate that the AmA1 intronic terminator sequence acts in a position-independent manner when placed within another gene.
Light regulated modulation of Z-box containing promoters by photoreceptor and downstream regulatory components, COP1 and HY5 in Arabidopsis thaliana
(Wiley-Blackwell, 2002) Yadav, Vandana; Kundu, Snehangshu; Chattopadhyay, Debasis; Negi, Prem; Wei, Ning; Deng, Xing-Wang; Chattopadhyay, Sudip
The Z-box is one of the light-responsive elements (LREs) found in the promoters of light inducible genes. We have studied the light responsive characteristics of Z-box containing synthetic as well as native promoters. We show that promoters with Z-box as a single LRE or paired with another LRE can respond to a broad spectrum of light. The response is primarily mediated by phyA, phyB and CRY1 photoreceptors at their respective wavelengths of light. We have demonstrated that CAB1 and Z-GATA containing promoters are down-regulated in hy5 mutants in the light. On the other hand, a promoter with Z-box alone is down-regulated in hy5 mutants both in dark and in light conditions, suggesting involvement of a similar regulatory system in the regulation of the promoter in two distinct developmental pathways: skotomorphogenesis and photomorphogenesis. Furthermore, similar to the CAB1 promoter, a Z-GATA containing promoter is derepressed in cop1 mutants in the dark. DNA±protein interaction studies reveal the presence of a DNA-binding activity that is speci®c to Z-box. These results provide insights into the regulation of the Z-box LRE mediated by various light signaling components.
Cloning and characterization of 5'-flanking region of oxalate decarboxylase gene from Flammulina velutipes
(Biochemical Society, 2002) Azam, Mohammad; Kesarwani, Meenu; Chakraborty, Subhra; Natarajan, Krishnamurthy; Datta, Asis
The oxalate-degrading enzyme, oxalate decarboxylase (OXDC), was purified and characterized from Flammulina elutipes, a basidiomycetous fungus [Mehta and Datta (1991) J. Biol. Chem. 266, 23548–23553]. The cDNA cloning and analyses revealed that OXDC transcription was induced by oxalic acid. However, in this report, we show that OXDC transcription is induced by low pH, not by oxalate. To understand the regulatory mechanism of OXDC expression, we have cloned and analysed a 580-bp genomic fragment from the 5h-flanking region of the OXDC gene. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs within the k580 bp of the upstream region. Electrophoretic-mobility-shift assays with partially purified cell extracts revealed specific binding of a factor in acid-induced, but not in uninduced, extracts. Furthermore, DNase I protection assays using the partially purified fraction from oxalic acid-induced extract revealed a footprint of a 13-bp sequence 5hGCGGGGTCGCCGA3h, termed low pH responsive element (LPRE), corresponding to the k287 to k275 bp region of the OXDC promoter. Our results suggest that in F. elutipes cells, activation of OXDC transcription in response to low pH is mediated by the binding of a novel transcription factor through the LPRE site in the OXDC promoter.
Active site geometry of oxalate decarboxylase from Flammulina velutipes: Role of histidine coordinated manganese in substrate recognition
(Cold Spring Harbor Laboratory Press, 2002) Chakraborty, Subhra; Chakraborty, Niranjan; Jain, Deepti; Salunke, Dinakara M; Datta, Asis
Oxalate decarboxylase (OXDC) from the wood-rotting fungus Flammulina velutipes, which catalyzes the conversion of oxalate to formic acid and CO2 in a single-step reaction, is a duplicated double-domain germin family enzyme. It has agricultural as well as therapeutic importance. We reported earlier the purification and molecular cloning of OXDC. Knowledge-based modeling of the enzyme reveals a ␤-barrel core in each of the two domains organized in the hexameric state. A cluster of three histidines suitably juxtaposed to coordinate a divalent metal ion exists in both the domains. Involvement of the two histidine clusters in the catalytic mechanism of the enzyme, possibly through coordination of a metal cofactor, has been hypoth- esized because all histidine knockout mutants showed total loss of decarboxylase activity. The atomic absorption spectroscopy analysis showed that OXDC contains Mn2+ at up to 2.5 atoms per subunit. Docking of the oxalate in the active site indicates a similar electrostatic environment around the substrate-binding site in the two domains. We suggest that the histidine coordinated manganese is critical for substrate recognition and is directly involved in the catalysis of the enzyme.
Light mediated regulation defines a minimal promoter region of TOP2
(Oxford University Press, 2003) Hettiarachchi, G. H. C. M.; Yadav, Vandana; Reddy, M. K.; Chattopadhyay, Sudip; Sopory, Sudhir K.
Light signaling has been demonstrated to be an important factor for plant growth and development; however, its role in the regulation of DNA replication and cell cycle has just started to be unraveled. In this work, we have demonstrated that the TOP2 promoter of Pisum sativum (pea) is activated by a broad spectrum of light including far-red light (FR), red light (RL) and blue light (BL). Deletion analyses of the TOP2 promoter in transformed plants, Arabidopsis thaliana and Nicotiana tobaccum (tobacco), de®ne a minimal promoter region that is induced by RL, FR and BL, and is essential and suficient for light-mediated activation. The minimal promoter of TOP2 follows the phytochromemediated low- ̄uence response similar to complex light regulated promoters. DNA±protein interaction studies reveal the presence of a DNA binding activity speci®c to a 106 bp region of the minimal promoter that is crucial for light-mediated activation. These results altogether indicate a direct involvement of light signaling in the regulation of expression of TOP2, one of the components of the DNA replication/cell cycle machinery.
Leader RNA binding ability of chandipura virus P protein is regulated by its phosphorylation status: A possible role in genome transcription-replication switch
(Elsevier B.V., 2003) Basak, Soumen; Raha, Tamal; Chattopadhyay, Debasis; Majumder, Amitabha; Shaila, M.S.; Chattopadhyay, D.J.
The molecular events associated with the transcriptive and replicative cycle of negative-stranded RNA viruses are still an enigma. We took Chandipura virus, a member of the Rhabdoviridae family, as our model system to demonstrate that Phosphoprotein P, besides Nucleocapsid protein N, also acts as a leader RNA-binding protein in its unphosphorylated form, whereas CKII-mediated phosphorylation totally abrogates its RNA-binding ability. However, interaction between P protein and leader RNA can be distinguished from N-mediated encapsidation of viral sequences. Furthermore, P protein bound to leader chain can successively recruit N protein on RNA while itself being replaced. We also observed that the accumulation of phosphorylation null mutant of P protein in cells results in enhanced genome RNA replication with concurrent increase in the viral yield. All these results led us to propose a model explaining viral transcription-replication switch where Phosphoprotein P acts as a modulator of genome transcription and replication by its ability to bind to the nascent leader RNA in its unphosphorylated form, promoting read-through of the transcription termination signals and initiating nucleocapsid assembly on the nascent RNA chain.
Isolation and characterization of sequence-tagged microsatellite sites markers in chickpea (Cicer arietinum L.)
(Wiley-Blackwell, 2003) Sethy, Niroj K.; Shokeen, Bhumika; Bhatia, Sabhyata
In this study we report the isolation of microsatellite sequences and their conversion to sequence-tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.
Salt-tolerant mutants in glycophytic salinity response (GSR) genes in Catharanthus roseus
(Springer, 2003) Rai, S.P.; Luthra, R.; Kumar, S.
The periwinkle Catharanthus roseus shares glycophytic properties of crop plants. To contribute towards an understanding of the glycophytic response to salinity, large populations of M2 seeds having an origin in nitroso-methyl urea and ethyl methane sulphonate treatments were screened for germination with 250 mM of NaCl. Out of the nine mutant lines so recovered, which tolerated salt stress due to loss of the normal glycophytic salinity response (GSR), the characteristics of six gsr mutants are reported here. All six, gsr-1 to gsr-6, differed from the wild-type in both seedling and adult- plant morphological characters beside being salt tolerant. The mutations in them were inherited as monogenic recessive alleles at the corresponding wild-type loci. The trans-complementation tests revealed that the gsr-1 to gsr-6 mutants specified six complementation groups. The mutant seedlings generally accumulated more proline and glycine betaine, constitutively, than the wild- type. The mutant plants transpired lower amounts of water and accumulated higher amounts of proline under drought stress. It was inferred that the products of the six GSR genes defined here are involved in the regulation of salt stress, as well as cell division, developmental and/or morphogenetic pathway(s), in C. roseus.